Neuritic Beading Induced by Activated Microglia Is an EarlyFeature of Neuronal Dysfunction Toward Neuronal Death byInhibition of Mitochondrial Respiration and Axonal Transport*

Received for publication, December 9, 2004, and in revised form, January 6, 2005Published, JBC Papers in Press, January 7, 2005, DOI 10.1074/jbc.M413863200

Hideyuki Takeuchi‡, Tetsuya Mizuno, Guiqin Zhang, Jinyan Wang, Jun Kawanokuchi,Reiko Kuno, and Akio Suzumura

From the Department of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University,Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

Recent studies suggest that excitotoxicity may con-tribute to neuronal damage in neurodegenerative dis-eases including Alzheimer disease, Parkinson disease,amyotrophic lateral sclerosis, and multiple sclerosis. Ac-tivated microglia have been observed around degenera-tive neurons in these diseases, and they are thought toact as effector cells in the degeneration of neural cells inthe central nervous system. Neuritic beading, focalbead-like swellings in the dendrites and axons, is a neu-ropathological sign in epilepsy, trauma, ischemia, aging,and neurodegenerative diseases. Previous reportsshowed that neuritic beading is induced by variousstimuli including glutamate or nitric oxide and is a neu-ronal response to harmful stimuli. However, the precisephysiologic significance of neuritic beading is unclear.We provide evidence that neuritic beading induced byactivated microglia is a feature of neuronal cell dysfunc-tion toward neuronal death, and the neurotoxicity ofactivated microglia is mediated through N-methyl-D-as-partate (NMDA) receptor signaling. Neuritic beading oc-curred concordant with a rapid drop in intracellularATP levels and preceded neuronal death. The actualneurite beads consisted of collapsed cytoskeletal pro-teins and motor proteins arising from impaired neuro-nal transport secondary to cellular energy loss. Thedrop in intracellular ATP levels was because of the in-hibition of mitochondrial respiratory chain complex IVactivity downstream of NMDA receptor signaling.Blockage of NMDA receptors nearly completely abro-gated mitochondrial dysfunction and neurotoxicity.Thus, neuritic beading induced by activated microgliaoccurs through NMDA receptor signaling and repre-sents neuronal cell dysfunction preceding neuronaldeath. Blockage of NMDA receptors may be an effectivetherapeutic approach for neurodegenerative diseases.

Central nervous system inflammation including microglialactivation likely contributes to the neurotoxicity observed inneurodegenerative diseases such as Alzheimer disease, Parkin-

son disease, amyotrophic lateral sclerosis, and multiple sclero-sis (1–7). Additionally, excitotoxicity might lead to neuronaldamage in these neurodegenerative diseases (8, 9). Microgliacan act as not only antigen-presenting cells but also effectorcells to damage central nervous system cells directly in vitroand in vivo (10–18). Conversely, microglia may have neuropro-tective effects mediated by neurotrophin release, glutamateuptake, and ingesting neurotoxic substances (19–22). There-fore, the role of microglia in either the pathogenesis of orprotection from neurodegenerative diseases is still entirelyunresolved.

Focal bead-like swelling in dendrites and axons (neuriticbeading) is thought to be a neuropathological sign in ischemia(23), epilepsy (24), mechanical pressure (25), brain tumor (26),aging (27), and neurodegenerative diseases such as Alzheimerdisease (28), Parkinson disease (29), and amyotrophic lateralsclerosis (30, 31). Neuritic beading is also induced by variousstimuli such as glutamate, nitric oxide (NO),1 hypoxia, oxida-tive stress, glucose starvation, and hypotonic conditions (32–38). Several previous studies reported that neuritic beadingwas a reversible response to neurotoxic stimuli independent ofneuronal death (32, 36). On the contrary, a recent study dem-onstrated that dendritic beading correlated with disease sever-ity in experimental autoimmune encephalomyelitis rat spinalcord (39), suggesting that beading paralleled neuronal damage.Furthermore, the mechanisms underlying neuritic bead forma-tion are completely unknown. Despite being a well documentedphenomenon, the pathological and functional significance ofneuritic beading are not known.

In this study, we sought to elucidate the mechanisms regu-lating the induction of neuritic beading by activated microglia.We show that neuritic beading induced by activated microgliais a feature of neuronal cell dysfunction preceding neuronaldeath, and this neurotoxicity is mediated by N-methyl-D-aspar-tate (NMDA) receptor signaling following glutamate binding.Blockade of the NMDA receptor may protect against the devel-opment of neurodegenerative diseases.

EXPERIMENTAL PROCEDURES

Cell Culture—All reagents except those specifically mentioned wereobtained from Sigma. The protocols for animal experiments were ap-proved by the Animal Experiment Committee of Nagoya University.* This work was supported by grants from the Ministry of Health,

Labor and Welfare of Japan, and a Center of Excellence grant from theMinistry of Education, Culture, Sports, Science and Technology of Ja-pan. The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

‡ To whom correspondence should be addressed: Dept. of Neuroim-munology, Research Institute of Environmental Medicine, Nagoya Uni-versity, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Tel.: 81-52-789-3883; Fax: 81-52-789-5047; E-mail: htake@riem.nagoya-u.ac.jp.

1 The abbreviations used are: NO, nitric oxide; NMDA, N-methyl-D-aspartate; TNF-�, tumor necrosis factor �; NOS, nitric-oxide synthe-tase; PI, propidium iodide; TUNEL, terminal deoxynucleotidyltrans-ferase-mediated UTP end labeling; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MAP2,microtubule-associated protein 2; NF, neurofilament; p-NF, phospho-rylated neurofilament; TMPD, N,N,N�,N�-tetramethyl-p-phenylenedia-mine; L-NMMA, NG-monomethyl-L-arginine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 11, Issue of March 18, pp. 10444–10454, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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Microglia were isolated from primary mixed glial cell cultures fromnewborn C57/BL6 mice on the 14th day with the “shaking off ” methodas described previously (40). The purity of the cultures was 97 to 100%as determined by Fc receptor-specific immunostaining as describedpreviously (40). Cultures were maintained in Dulbecco’s modified Ea-gle’s minimum essential medium supplemented with 10% fetal calfserum (JRH Biosciences, Lenexa, KS), 5 �g/ml bovine insulin, and 0.2%glucose. Neuron cultures were prepared from C57BL/6 mice at embry-onic day 17 using the Nerve-Cell Culture System (Sumitomo Bakelite,Akita, Japan) as described previously (41–43). Briefly, cortices weredissected and freed of meninges. Cortical fragments were dissociatedinto single cells using dissociation solution, and they were resuspendedin Nerve-Cell Culture Medium (serum-free conditioned medium from48-h rat astrocyte confluent cultures based on Dulbecco’s modifiedEagle’s minimum essential medium/F-12 with N2 supplement, Sumi-tomo Bakelite). Primary neuronal cells were plated on 12-mm polyeth-yleneimine-coated coverslips (Asahi Techno Glass Corp., Chiba, Japan)in 24-well multidishes at a density of 5 � 104 cells/well. The purity ofthe cultures was more than 95% as determined by NeuN-specific im-munostaining as described previously (42, 43).

To activate microglia, 5 � 104 microglia were plated on 24-wellmultidishes with Nerve-Cell Culture Medium (Sumitomo Bakelite) con-taining 1 �g/ml lipopolysaccharide and 100 ng/ml interferon-� (R&DSystems, Minneapolis, MN). The pan-nitric-oxide synthetase (NOS)inhibitor NG-monomethyl-L-arginine (L-NMMA, Calbiochem, San Di-ego, CA) at a final concentration of 1 mM was added when the inhibitionof microglial inducible NOS was needed. After a 16-h incubation, acti-vated microglia-conditioned medium was applied to each well contain-ing 5 � 104 neurons at 10–13 days in vitro. Assessments were per-formed at each time point (0, 1, 3, 6, 12, and 24 h) after mediumexchange. Neurons were preincubated with the indicated drug for 1 h.Neurons were then incubated with activated microglia-conditioned me-dium containing each drug. The final concentrations of each drug wereas follows: pan-NOS inhibitor, 1 mM L-NMMA; NO and ONOO� scav-enger, 50 �M 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (Biomol, Plymouth Meeting, PA); ONOO� scavenger, 10�M 5,10,15,20-tetrakis(N-methy-4�-pyridyl)porphinato iron(III) (Calbio-chem); NMDA receptor antagonist, 10 �M MK801 (Calbiochem); non-NMDA receptor antagonist, 20 �M 6-cyano-7-nitroquinoxaline-2,3-dione;neutralizing antibody for tumor necrosis factor (TNF)-�, 0.1 mg/mlanti-mouse TNF-� antibody (R&D Systems); pan-caspase inhibitor, 20mM z-VAD-fmk (Peptide Institute, Osaka, Japan); Ca2� chelator, 1 mM

EDTA; tubulin polymerization stabilizer, 10 �M Taxol; actin polymeri-zation stabilizer, 10 �M phalloidin. To examine NMDA neurotoxicity,100 �M NMDA was added to neurons.

Assessment of Neuritic Beading—To assess neuritic beading, neuronswere observed under a phase-contrast microscope at each time point (0,1, 3, 6, 12, and 24 h) by a modification of the method previously reportedby Park et al. (32). More than 200 neurons in duplicate wells wereassessed blindly in three independent trials. The ratio of bead-bearingneurons was calculated as a percentage of total cells.

Assessment of Cell Death—Cell death was assessed by the dye-exclu-sion method with propidium iodide (PI; Molecular Probes, Eugene, OR)as described previously (44). At each time point (0, 1, 3, 6, 12, and 24 h)after stimulation, cells were incubated with 2 �g/ml PI-containingmedium for 15 min at 37 °C. More than 200 neurons in duplicate wellswere assessed blindly in three independent trials under a conventionalfluorescent microscope. The ratio of dead cells was calculated as apercentage of PI-positive cells among total cells.

To detect apoptosis, we used the terminal deoxynucleotidyltrans-ferase-mediated UTP end labeling (TUNEL) assay with the in situ celldeath detection kit (Roche Diagnostics) as described previously (44).TUNEL assay was carried out at each time point (0, 1, 3, 6, 12, and 24 h)according to the manufacturer’s protocol. As a positive control, neuronswere incubated with 10 nM staurosporin for 24 h. More than 200neurons in duplicate wells were assessed blindly in three independenttrials under a conventional fluorescent microscope. The ratio of apop-totic cells was calculated as a percentage of TUNEL-positive cellsamong total cells.

Assessment of Intracellular ATP Levels—To measure intracellularATP levels, we used a luminometric assay with ApoSENSOR CellViability Assay Kit (BioVision, Mountain View, CA) according to themanufacturer’s protocol. Assays were carried out at each time point (0,1, 3, 6, 12, and 24 h) in six independent trials. ATP concentration ateach time point was calculated as a percentage of control.

Assessment of Mitochondrial Respiration Inhibition—To analyze mi-tochondrial respiration inhibition, we carried out a mitochondrial res-piration recovery assay by modifying the method previously reported by

Rego et al. (45). If complex I is inhibited, mitochondrial respirationrecovers following the addition of succinate as a substrate for complexII/III. If complex II/III are inhibited, mitochondrial respiration recoversafter the addition of ascorbic acid and TMPD as substrates for complexIV. If complex IV is inhibited, mitochondrial respiration does not re-cover following the addition of these drugs. Neurons were preincubatedfor 1 h with each drug, and neurons were then incubated with activatedmicroglia-conditioned medium containing drug. The final concentrationof each drug was as follows: 5 mM succinate, 5 mM ascorbic acid, and0.25 mM TMPD. 10 �M Rotenone was added to inhibit complex I activity.0.25 �g/ml Antimycin A was added to inhibit complex II/III activity. 0.5mM NaN3 was added to inhibit complex IV activity. Recovery of mito-chondrial respiration was detected by measuring the intracellular ATPlevels. Assays were carried out after a 3-h incubation in six independenttrials. Each ATP concentration was calculated as a percentageof control.

Assessment of Mitochondrial Impairment—To assess mitochondrialmembrane potential, we used MitoTracker Red CMXRos (MolecularProbes), a dye whose staining intensity is directly proportional to mi-tochondrial membrane potential at concentrations lower than 50 nM. Ateach time point (0, 1, 3, 6, 12, and 24 h), cells were incubated with 20 nM

MitoTracker-containing medium for 30 min at 37 °C. Cells were fixedand permeabilized as described below under “Immunocytochemistry.”Fluorescent signal intensity was quantified with a confocal laser scan-ning microscopic system (LSM510; Carl Zeiss, Oberkochen, Germany).More than 100 cells in duplicate wells were assessed blindly in threeindependent trials. Relative signal intensity at each time point wasexpressed as a percentage of control.

To assess mitochondrial viability, we used the 3-(4,5-dimethylthia-zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay with CellTiter 96 Aqueous One solution assay (Promega,Madison, WI) according to the manufacturer’s protocol as describedpreviously (44). Absorbance at 490 nm was measured in a multiple platereader. Assays were carried out at each time point (0, 1, 3, 6, 12, and24 h) in six independent trials.

Assessment of NO Generation—To measure NO production, we per-formed the Griess reaction on the media at each time point (0, 1, 3, 6, 12,and 24 h) as described previously (16). Absorbance at 540 nm wasmeasured in a multiple plate reader. Assays were carried out in sixindependent trials.

Assessment of Glutamate Release—To measure extracellular gluta-mate concentrations, we used the Glutamate Assay Kit colorimetricassay (Yamasa Corp., Tokyo, Japan) according to the manufacturer’sprotocol at each time point (0, 1, 3, 6, 12, and 24 h). Absorbance at 600nm was measured in a multiple plate reader. Assays were carried out insix independent trials.

Immunocytochemistry—At each time point (0, 1, 3, 6, 12, and 24 h),neurons were fixed with 4% paraformaldehyde for 30 min and perme-abilized with 0.05% Triton X-100 for 10 min at room temperature. Cellswere stained with the primary antibody at 4 °C overnight as follows:mouse monoclonal anti-neuron specific tubulin �III isoform (�III-tubu-lin) antibody (1:2,000, Chemicon International, Temecula, CA), mousemonoclonal anti-microtubule-associated protein 2 (MAP2) antibody (1:500, Chemicon International), mouse monoclonal anti-phosphorylatedneurofilament (p-NF) antibody (SMI31, 1:5,000, Sternberger Monoclo-nus Inc., Lutherville, MD), rabbit polyclonal anti-manganese superox-ide dismutase (MnSOD) antibody (1:2,000, Stressgen Biotechnologies,Victoria, BC, Canada), mouse monoclonal anti-kinesin antibody (1:1,000, Chemicon International), and mouse monoclonal anti-cytoplas-mic dynein antibody (1:100, Chemicon International). They were sub-sequently stained with secondary antibody-conjugated Alexa-488, -568,or -647 (1:1,000, Molecular Probes) at room temperature for 90 min.

FIG. 1. Activated microglia induce neuritic beading. Imageswere taken under a phase-contrast microscope. A, control neurons. B,neurons incubated with activated microglia-conditioned medium for6 h. Control neurons bore few neuritic beads (A). In contrast, activatedmicroglia-conditioned medium induced numerous neuritic beads (B,arrows) accompanied by neurite narrowing. Scale bar, 10 �m.

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FIG. 2. A rapid increase in neuritic beading and a rapid drop in intracellular ATP levels because of inhibition of mitochondrialrespiration are early features of neuronal cell dysfunction by activated microglia. A, frequency of bead-bearing cells. E, control; ●,neurons with activated microglia-conditioned medium. B, frequency of dead cells. E, PI-positive neurons in control; ●, PI-positive neurons withactivated microglia-conditioned medium; �, TUNEL-positive neurons in control; f, TUNEL-positive neurons with activated microglia-conditionedmedium. C, MTS assay. D, intracellular ATP level. E, fluorescent signal intensity of MitoTracker. Note the rapid increase in bead-containing cellsand the rapid drop in intracellular ATP level. Cell viability and mitochondrial function were relatively spared during an early period (shaded area).

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Cells were then counterstained with 1 �g/ml Hoechst 33342 (MolecularProbes) at room temperature for 10 min, and mounted in antifadereagent. Cells were analyzed under a confocal laser-scanning micro-scope (LSM510, Carl Zeiss).

Analysis of Axonal Transport—Axonal transport was analyzed un-der a time-lapse phase-contrast microscopic system (Axiovert 200M/Cell Observer System, Carl Zeiss). The number of particles (diameter�50 nm) moving antegrade and retrograde in axons were counted for eachhour (0�6 h) as described previously (46). Three independent fields wereassessed blindly. Data were expressed as a percentage of control.

Statistical Analysis—All results were analyzed by one-way analysisof variance with a Tukey-Kramer post-hoc test using Statview softwareversion 5 (SAS Institute Inc., Cary, NC).

RESULTS

Activated Microglia Induce Neuritic Beading and Subse-quent Neuronal Cell Death—Activated microglia conditionedmedium rapidly induced numerous beads in most neurites(Figs. 1B and 2A). Beaded neurites were narrow and palecompared with control cells, reflecting neurite damage (Fig.1B). Activated microglia-conditioned medium also induced neu-ronal cell death at a later phase (Fig. 2B), but very littleapoptotic cell death was detected by TUNEL assay (Fig. 2B).Thus, activated microglia appear to induce necrotic cell deathin neurons; this is consistent with previous reports (47, 48).

FIG. 3. Neuritic beads colocalized with collapsed cytoskeletal proteins. Time course images were taken under a confocal laser-scanningmicroscope. Neurons were stained with mouse monoclonal anti-�III-tubulin antibody (green, A–F), mouse monoclonal anti-phosphorylated NF(p-NF) antibody (SMI31, red, G–L), and mouse monoclonal anti-MAP2 antibody (blue, M–R). Neuritic beads colocalized with �III-tubulin, p-NF, andMAP2 (T–X and arrows in Y). Y is an enlargement of U. Note the gradual collapse and decrease in immunoreactivity of cytoskeletal proteins. Thedensity of the neuronal network decreased with increasing neuronal loss. Scale bar, 10 �m.

F, mitochondrial respiration recovery assay. cont, control; NMDA, control with 100 �M NMDA; Mi, neurons with activated microglia-conditionedmedium; cont � rot, control with 10 �M rotenone; cont � rot � suc, control with 10 �M rotenone and 5 mM succinate; NMDA � suc, control with100 �M NMDA and 5 mM succinate; Mi � suc, neurons with activated microglia-conditioned medium and 5 mM succinate; cont � ant, control with0.25 �g/ml antimycin A; cont � asc � TMPD, control with 5 mM ascorbic acid and 0.25 mM TMPD; NMDA � asc � TMPD, control with 100 �M

NMDA, 5 mM ascorbic acid, and 0.25 mM TMPD; Mi � asc � TMPD, neurons with activated microglia-conditioned medium, 5 mM ascorbic acid,and 0.25 mM TMPD; cont � NaN3, control with 0.5 mM NaN3; cont � NaN3 � all sub, control with 0.5 mM NaN3, 5 mM succinate, 5 mM ascorbicacid, and 0.25 mM TMPD; NMDA � all sub, control with 100 �M NMDA, 5 mM succinate, 5 mM ascorbic acid, and 0.25 mM TMPD; Mi � all sub,neurons with activated microglia-conditioned medium, 5 mM succinate, 5 mM ascorbic acid, and 0.25 mM TMPD. *, p � 0.05 versus control. Valuesare mean � S.D.

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A Rapid Increase in Neuritic Beading and a Rapid Drop inIntracellular ATP Levels Are Early Features of Neuronal CellDysfunction Induced by Microglia—Activated microglia-condi-tioned medium induced a rapid increase in bead-containingneurons (Fig. 2A) and a rapid drop in intracellular ATP levels(Fig. 2D), whereas cell viability (Fig. 2B), mitochondrial reduc-tion activity (Fig. 2C), and mitochondrial membrane potential(Fig. 2E) were relatively unchanged during the early stagesafter treatment (Fig. 2, shaded areas).

During this early period, neurons were likely running anenergy deficit to maintain homeostasis. Neuritic beading couldbe an early feature of neuronal cell dysfunction resulting fromenergy loss induced by activated microglia.

Activated Microglia Inhibit Mitochondrial Respiratory ChainComplex IV Activity in Neurons—We carried out mitochondrialrespiration recovery assays to determine the mitochondrialrespiratory chain complex in neurons that are inhibited byactivated microglia (Fig. 2F). Rotenone, a complex I inhibitor,reduced intracellular ATP levels by �80% that seen in controls,and this reduction was abrogated by the addition of succinate,a substrate of complex II/III. Antimycin A, a complex II/III

inhibitor, reduced intracellular ATP levels by �85% comparedwith controls, and ascorbic acid and TMPD, complex IV sub-strates, reversed these effects. NaN3, a specific complex IVinhibitor, led to a reduction in intracellular ATP levels of �90%compared with controls, and no substrates were able to restorenormal ATP levels. As seen with NaN3, no substrates were ableto restore intracellular ATP levels or mitochondrial respirationof neurons treated with activated microglia (Fig. 2F, blackcolumns). Thus, activated microglia inhibit neuronal mitochon-drial respiration primarily through inhibiting complex IV ac-tivity. We also confirmed that NMDA inhibited complex IVactivity (Fig. 2F, dotted columns).

Neuritic Beads Colocalized with Collapsing Cytoskeletal Pro-teins—Time course immunocytochemical analysis revealedthat neuritic bead formation was accompanied by the gradualcollapse of the neuronal network (Fig. 3). Neuritic beads colo-calized with tubulin (Fig. 3, A–F and Y), p-NF (Fig. 3, G–L andY), and MAP2 (Fig. 3, M–R and Y). It was unclear whetherneuritic beads colocalized with actin (data not shown). Thefluorescent signal arising from these cytoskeletal proteinsgradually weakened as the neuronal network was collapsing.

FIG. 4. Time course analysis of neuronal mitochondria. Time course images were taken under a confocal laser-scanning microscope.Neurons were stained with rabbit polyclonal anti-MnSOD antibody (a mitochondrial marker, green, A–F), MitoTracker Red CMXRos (red, G–L),and mouse monoclonal anti-�III-tubulin antibody (blue, M–R). The mitochondria were first distributed throughout the neuronal network. Y and Zare enlargements of S and X, respectively. After addition of activated microglia-conditioned medium, the mitochondria gradually accumulated inthe cell body and disappeared from distal neurites (A–F). Mitochondria morphology also changed from a granular pattern to a tubular and reticularpattern (A–F, Y, and Z). Note the sparing of MitoTracker fluorescence during an early stage following treatment (those quantitative data wereindicated in Fig. 2E). Scale bar, 10 �m.

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Neuritic bead formation is associated with the collapse of cy-toskeletal proteins such as tubulin, NF, and MAPs.

Activated Microglia Affect the Morphology and Distributionof Neuronal Mitochondria—Time course immunocytochemicalanalysis also revealed that activated microglia-conditioned me-dium changed the morphology and distribution of mitochondriain neurons (Fig. 4). Prior to treatment, mitochondria weredistributed throughout the neurites and cell bodies (Fig. 4, Aand S). After the addition of activated microglia-conditionedmedium, however, the mitochondria gradually collected in thecell body and disappeared from distal neurites (Fig. 4, B–F andT–X) as the neuronal network was collapsing (Fig. 4, N–R). Themitochondria partially colocalized with proximal neuritic beadsbecause they disappeared from distal neurites. As the distri-bution of the mitochondria changed, their morphology shiftedgradually from a granular pattern to a tubular and reticularpattern (Fig. 4, A–F, Y, and Z), reflecting a shift in the demandfor mitochondrial energy (49).

As shown in Fig. 2E, the mitochondrial membrane potentialwas relatively unchanged during the early stages followingtreatment with activated microglia-conditioned medium (Fig.4, G–I), but it gradually declined over time (Fig. 4, J–L) asassessed by the fluorescence intensity of the MitoTracker dye.

Neuritic Beads Colocalized with Collapsed Axonal TransportMotor Proteins—We next examined the effects of microglia on

the motor proteins responsible for axonal transport becauseactivated microglia disturbed the cytoskeletal network andmitochondrial distribution in neurons. Time course immunocy-tochemical analysis revealed that neuritic beads colocalizedwith kinesin, a motor protein involved in antegrade fast axonaltransport (Fig. 5), and cytoplasmic dynein, a motor proteinactive in retrograde fast axonal transport (Fig. 6).

Initially, kinesin was distributed throughout the neuronalnetwork (Fig. 5, A and S). After addition of activated microglia-conditioned medium, however, kinesin strongly accumulatedalong the neurites in a pattern consistent with aggregation.These accumulations colocalized with sites of neuritic beading(Fig. 5, B–F and T–X and Y). The immunoreactivity of kinesinin the cell body also decreased with time.

Like kinesin, cytoplasmic dynein was initially distributedthroughout the neuronal network (Fig. 6, A and S). After theaddition of activated microglia-conditioned medium, cytoplasmicdynein immunoreactivity decreased gradually in the neurites.Cytoplasmic dynein accumulated in an aggregate pattern alongthe neurites to a lesser extent than kinesin with similar kinetics.These sites also colocalized with neuritic beads (Fig. 6, B–F, T–X,and Y). The immunoreactivities of cytoplasmic dynein in theproximal neurite and cell body were relatively unaffected.

Taken together, these data suggest that activated microgliaimpair kinesin function to a greater extent than cytoplasmic

FIG. 5. Collapsed kinesin accumulates in neuritic beads. Time course images were taken under a confocal laser-scanning microscope.Neurons were stained with mouse monoclonal anti-kinesin antibody (green, A–F), mouse monoclonal anti-phosphorylated NF (p-NF) antibody(SMI31, red, G–L), and mouse monoclonal anti-MAP2 antibody (blue, M–R). Y is an enlargement of U. Kinesin was first distributed throughoutthe neuronal network. After addition of activated microglia-conditioned medium, it accumulated strongly along the neurites in an aggregatepattern that colocalized with neuritic beads (T–X and arrows in Y). Simultaneously, its immunoreactivity decreased in the cell body. Scale bar,10 �m.

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dynein. This implies that microglia selectively inhibit fast ax-onal transport, and we further investigated this hypothesis.

Activated Microglia Inhibit Fast Axonal Transport—Time-lapse phase-contrast imaging revealed that activated microglia-conditioned medium inhibited fast axonal transport (Fig. 7). Ad-ditionally, the decreased levels of intracellular ATP induced byactivated microglia inhibited axonal transport. The inhibition oftransport led to the focal accumulations of motor proteins andcytoskeletal proteins such as kinesin, cytoplasmic dynein, tubu-lin, NF, and MAPs, that become apparent as neuritic beads.

Interestingly, retrograde transport was affected to a lesserextent (1–3 h) than antegrade transport during the earlyphases of this response; this was consistent with the immuno-cytochemical observations of kinesin and cytoplasmic dyneindescribed above. We further hypothesize that the asymmetricinhibition of axonal transport leads to the accumulation ofmitochondria at the neuronal cell body that is observed afteraddition of activated microglia-conditioned medium.

Activated Microglial Neurotoxicity Is Primarily Mediated byReleased Glutamate through NMDA Receptor Signaling—Acti-vated microglia-conditioned medium had high concentrationsof NO (Fig. 8A) and glutamate (Fig. 8B). In addition to NO andglutamate, activated microglia/macrophage release inflamma-tory cytokines such as interleukin-1�, interleukin-6, interfer-on-�, and TNF-� (42, 50–54). We confirmed that recombinantcytokines did not induce significant cell death except for TNF-

�.2 Moreover, recent studies reported that neuronal death inneurodegenerative diseases might be a non-apoptotic but

2 H. Takeuchi, T. Mizuno, G. Zhang, J. Wang, J. Kawanokuchi, R.Kuno, and A. Suzumura, unpublished data.

FIG. 6. Collapsed cytoplasmic dynein accumulates in neuritic beads. Time course images were taken under a confocal laser-scanningmicroscope. Neurons were stained with mouse monoclonal anti- cytoplasmic dynein antibody (green, A–F), mouse monoclonal anti-phosphorylatedNF (p-NF) antibody (SMI31, red, G–L), and mouse monoclonal anti-MAP2 antibody (blue, M–R). Y is an enlargement of V. Cytoplasmic dynein wasfirst distributed throughout the neuronal network, then its immunoreactivity in the neurites decreased gradually. Simultaneously, a mildaccumulation of cytoplasmic dynein in aggregates was observed along the neurites, and this colocalized with neuritic beads (T–X and arrows in Y).Its immunoreactivity in the cell body was relatively spared. Scale bar, 10 �m.

FIG. 7. Activated microglia inhibit fast axonal transport. Timelapse phase-contrast imaging revealed that activated microglia-condi-tioned medium inhibited fast axonal transport. ●, antegrade; f, retro-grade. Retrograde transport was affected to the lesser extent during anearly period following treatment (1–3 h, †) than antegrade transport. *,p � 0.05 versus control. †, p � 0.05 versus antegrade transport. Valuesare mean � S.D.

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caspase-dependent form of programmed cell death (44, 55–57).Thus, we evaluated the effects of drugs that affect NO, gluta-mate, TNF-�, and caspases.

The pan-NOS inhibitor L-NMMA completely inhibited micro-glial NO production when it was added to microglia at the same

time as stimulation (Fig. 8A, Mi L-NMMA). Modulation of neu-ronal NOS activity (activation with NMDA or inhibition withL-NMMA) did not affect the extracellular NO concentration(Fig. 8A, NMDA and Neu L-NMMA); our method was not suf-ficiently sensitive to detect the changes induced by neuronalNOS. NO scavengers, carboxy-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and 5,10,15,20-tetrakis(N-methy-4�-pyridyl)porphinato iron(III), signifi-cantly increased the concentration of nitrites in the samples,reflecting their reduction of NO (Fig. 8A). In contrast, neitherof these drugs affected glutamate release from activatedmicroglia (Fig. 8B).

NMDA induced neuritic beading and subsequent neuronaldeath markedly (Fig. 8, C and D, NMDA). Complete inhibitionof microglial NO release did not prevent neuritic beading andneuronal death (Fig. 8, C and D, Mi L-NMMA). In contrast,inhibiting endogenous NO production in neurons partially re-duced neuritic beading and cell death (Fig. 8, C and D, NeuL-NMMA). Blockage of NMDA receptors with MK801 dramat-ically ameliorated neuritic beading and cell death (Figs. 8, Cand D, and 9C). No effect was seen when cells were incubatedwith a non-NMDA inhibitor (Fig. 8, C and D, CNQX). Ca2�

influx through glutamate receptors is thought to lead to exci-totoxic neuronal death (47, 48, 58, 59). We evaluated the effectof the Ca2� chelator EDTA on neurite viability, and, surpris-ingly, it increased cell death (Fig. 8, C and D). Neither TNF-�neutralization (Fig. 8, C and D, anti-TNF) nor a pan-caspaseinhibitor (Fig. 8, C and D, zVAD-fmk) affected neuritic beadingor cell death. These data suggest that a programmed cell deathpathway is not responsible for neuritic beading and neuronaldeath induced by activated microglia; this is consistent withthe TUNEL assay data shown in Fig. 2B. We next assessed theeffects of drugs that stabilize cytoskeletal protein polymeriza-tion on cell death, but both Taxol and phalloidin increased celldeath (Fig. 8, C and D).

Blockage of NMDA Receptors Completely Rescues Neuronsfrom Activated Microglial Neurotoxicity—Because MK801nearly completely ameliorated neuritic beading and neuronaldeath induced by activated microglia, we assessed its effects onmitochondrial function and axonal transport. MK801 rescuedneurons from mitochondrial impairment (Fig. 9D), intracellu-lar ATP decreases (Fig. 9E), and axonal transport damage (Fig.9F) induced by activated microglia. These data clearly linkactivated microglial neurotoxicity, including neuritic beadingand cell death, to glutamate excitotoxicity downstream ofNMDA receptor signaling.

DISCUSSION

Here we provide evidence that neuritic beading induced byactivated microglia correlates with neuronal cell dysfunctionand precedes neuronal death. Additionally, microglial neuro-toxicity is primarily mediated by NMDA receptor signalingfollowing ligation of released glutamate. Neuritic bead forma-tion was accompanied by a drop in intracellular ATP levels,and it preceded neuronal death. The observed drop in intracel-lular ATP levels was because of inhibition of the mitochondrialrespiratory chain complex IV activity, and the loss of intracel-lular energy pools negatively affected neuronal transport. Im-paired transport caused cytoskeletal and motor protein accu-mulation at sites of neuritic beading. Finally, blockage ofNMDA receptors abrogated all the observed signs of micro-glial neurotoxicity including neuritic bead formation, mito-chondrial impairment, neuronal transport damage, and sub-sequent neuronal death. Taken together, the neuritic beadsinduced by activated microglia are thought to consist of theresidual cargo of neuronal transport vesicles that accumulate

FIG. 8. Assessment of drug effects. Assessments were performed24 h after medium change. A, drug effect on NO concentration. B, drugeffect on glutamate concentration. No drug affected glutamate concen-tration in the medium. C, drug effect on neuritic beading. D, drug effecton neuritic beading and cell death. NMDA, control with 100 �M NMDA;Mi, neurons with activated microglia-conditioned medium; MiL-NMMA, neurons with activated microglia-conditioned medium in-cluding L-NMMA, which added simultaneously induced stimulation toactivate microglia (to inhibit microglial inducible NOS (iNOS)); NeuL-NMMA, neurons with activated microglia-conditioned medium andL-NMMA added 1 h before medium exchange (to inhibit neuronal NOS).*, p � 0.05 versus control. †, p � 0.05 versus Mi. The values are themean � S.D. PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazo-line-1-oxyl-3-oxide; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;FeTMPyP, 5,10,15,20-tetrakis(N-methy-4�-pyridyl)porphinato iron(III).

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following energy starvation downstream of NMDA receptorsignaling. Furthermore, our data demonstrate that neuronsdo not undergo apoptosis or another form of caspase-depend-ent cell death.

Neuritic beading is observed in various pathological condi-tions (23–31). Our current results indicate that neuritic beadsrepresent the accumulation of vesicular cargo and transportproteins following damage to the transport mechanisms byactivated microglia. Additionally, the neuritic beading ob-served in diseased brain tissue likely arises during a period ofrelative cellular energy deficit. Activated microglia releaselarge amounts of glutamate. Following glutamate ligation ofthe NMDA receptor, neuronal mitochondrial respiration wasinhibited and this led to neuronal energy loss. Neuronal trans-port is an extremely energy intense process, and we observedgross defects in the activity of microtubule-associated ATPasemotor proteins such as kinesin and cytoplasmic dynein. Thesemotor proteins, as well as cytoskeletal proteins and organelles

accumulated along the neurites because of impaired neuronaltransport, and these accumulations formed neuritic beads.Time-lapse imaging showed that particles sequestrated in neu-ritic beads were rotating like a top inside the bead (data notshown). Interestingly, retrograde transport was affected to alesser extent than antegrade transport during the early phaseafter treatment (Fig. 8). This was consistent with the immuno-cytochemical observations of kinesin and cytoplasmic dynein(Figs. 6 and 7). The mitochondria accumulated at the neuronalcell body and disappeared from distal neurites after the addi-tion of activated microglia-conditioned medium (Fig. 5). Duringperiods of energy stress, neurons retrieve mitochondria fromthe distal neurites to the cell body, and this process relies onretrograde axonal transport (60, 61). Thus, the preferentialmaintenance of retrograde transport following exposure to ac-tivated microglial-conditioned medium is consistent with thishypothesis. The morphology of mitochondria changed from agranular pattern to a tubular and reticular pattern as they

FIG. 9. Blockage of NMDA receptorscompletely rescues neurons from ac-tivated microglial neurotoxicity. A–C,images were taken under a phase-con-trast microscope. Scale bar, 10 �m. A,control neurons. B, neurons incubatedwith activated microglia-conditioned me-dium for 6 h. C, neurons incubated withactivated microglia-conditioned mediumincluding MK801 for 6 h. D, MTS assay.●, neurons with activated microglia-con-ditioned medium; E, neurons incubatedwith activated microglia-conditioned me-dium including MK801. E, intracellularATP levels. ●, neurons with activated mi-croglia-conditioned medium; E, neuronsincubated with activated microglia-condi-tioned medium including MK801. F, as-sessment of fast axonal transport. ● , an-tegrade transport in neurons withactivated microglia-conditioned medi-um; f, retrograde transport in neuronsincubated with activated microglia-con-ditioned medium; E, antegrade trans-port in neurons incubated with activatedmicroglia-conditioned medium includingMK801; �, retrograde transport in neu-rons incubated with activated microglia-conditioned medium including MK801.*, p � 0.05 versus control. †, p � 0.05versus antegrade transport in neuronswith activated microglia-conditioned me-dium. Values are mean � S.D.

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accumulated at the neuronal cell body (Fig. 5). Alterations inmitochondrial shape and structure are known to correlate withincreasing energy demands, and mitochondria with this mor-phology are frequently found in tumor cells (49). The changesin mitochondrial morphology induced by activated microgliaare further evidence of the neuronal energy deficit produced bythese cells.

Glutamate is known to cause neuronal damage throughexcitotoxicity (8, 9). In particular, the NMDA receptor path-way has been extensively studied in this regard. According toprevious studies, a Ca2� influx through NMDA receptor ac-tivates neuronal NOS and endogenous NO elevation leads tomitochondrial respiratory inhibition and direct neurotoxicity(58, 59). We confirmed that a high dose of NMDA decreasedrespiratory chain complex IV activity and induced neuriticbeading followed by neuronal death (Figs. 2F and 8, A–D). Inour study, the NMDA receptor antagonist MK801 nearlycompletely blocked the neurotoxicity induced by activatedmicroglia (Fig. 9). However, the NOS inhibitor L-NMMA onlypartially prevented microglial neurotoxicity despite its com-plete inhibition of NOS (Fig. 9). Direct inhibition of respira-tory chain complex IV activity by NaN3 also induced neuriticbeading and subsequent neuronal death (data not shown),which was in accordance with a previous report (62). Takentogether, in addition to NO, another unknown substanceinduced downstream of NMDA receptor signaling mayparticipate in the inhibition of respiratory chain complexIV activity. Further investigations are needed to clarifythis.

Controversy surrounds the issue of whether excitotoxic neu-ronal cell death through NMDA receptor is mediated by Ca2�

(47, 48, 58, 59) or Na� (34, 36). In our study, the Ca2� chelatorEDTA significantly increased neuronal death (Fig. 9). Clearly,EDTA might disturb intracellular Ca2� homeostasis, and morethorough studies are needed to address this question. Thepresent study did not provide any positive evidence that Ca2�

influx mediates the neuronal death signal through the NMDAreceptor.

Previous reports suggested that a caspase-dependent non-apoptotic form of cell death took place in neurodegenerativediseases (51, 52). However, treatment with the broad caspaseinhibitor z-VAD-fmk did not affect neuronal death in our study(Fig. 9). We propose that excitotoxic neuronal death throughthe NMDA receptor is much more like necrosis, not a pro-grammed cell death.

In this study, we assessed the effect of drugs that stabilizecytoskeletal protein polymerization. However, both the tubulinpolymerization stabilizer Taxol and the actin polymerizationstabilizer phalloidin increased neuronal death (Fig. 9). Theseobservations implied that collapse of the cytoskeleton per sewas only a feature of neuronal cell dysfunction, not a cause ofneuronal death. Our study suggested that either blockage ofNMDA receptors or an increase in intracellular ATP levels wasneeded to avoid neuronal cell death induced by activatemicroglia.

In conclusion, we demonstrated that neuritic beading in-duced by activated microglia was a feature of neuronal celldysfunction toward neuronal death downstream of NMDA re-ceptor signaling. Blockage of NMDA receptor may be an effec-tive strategy for the treatment of neurodegenerative diseasesincluding Alzheimers disease, Parkinsons disease, amyotro-phic lateral sclerosis, and multiple sclerosis.

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Reiko Kuno and Akio SuzumuraHideyuki Takeuchi, Tetsuya Mizuno, Guiqin Zhang, Jinyan Wang, Jun Kawanokuchi,

and Axonal TransportDysfunction Toward Neuronal Death by Inhibition of Mitochondrial Respiration

Neuritic Beading Induced by Activated Microglia Is an Early Feature of Neuronal

doi: 10.1074/jbc.M413863200 originally published online January 7, 20052005, 280:10444-10454.J. Biol. Chem. 

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